Self-renewal of the intestinal epithelium requires layers of precise control that are critical for tissue homeostasis. The epithelial lining of the intestine, a prime example of a homeostatic system, completely renews itself every 5 days. Amazingly, in response to continuous mechanical, chemical and pathogen-derived wounding, intestinal stems cells produce multiple cell types at just the right ratios throughout the entire lfe of the organism. Lack of this exquisite homeostatic control is the basis for a variety of disease including Crohn's disease, Irritable Bowel Syndrome, Ulcerative Colitis, and gastrointestinal cancers. Our understanding of how this complex epithelial system maintains such robust organization and how is goes wrong in disease is woefully incomplete. Recent studies have shown that intestinal epithelial cells are capable of a surprising degree of proliferative and self organizing behavior in 3-demensional cultures in vitro. In particular, mouse small intestinal stem cells give rise to ensembles of cells in stereotyped lineages, accurately polarized into tissue-lik structures. My goal is to identify and understand control mechanism of intestinal homeostasis through single cell and chemical approaches. I have developed the 2-D and 3-D culture of non-transformed mouse and human intestinal epithelium in high-throughput format. This allows me to utilize the unique single-cell profiling and analysis platform developed by our lab to study mechanism of gut homeostasis with a level of speed and cellular resolution never before achieved. Protein kinases are well known to play prominent roles in much of normal and pathological cell physiology. A wealth of information is known about individual kinase at the molecular and structural level, yet their role in how complex tissues, such as intestinal epithelium, renew and self-organize is unclear. My hypothesis is that key kinase networks modulate tissue homeostasis and therapeutic response. This Research Plan seeks to decipher how the kinase networks regulates gut homeostasis (Aim 1), how a central kinase hub, GSK-3, controls cell fate (Aim 2), and identify novel approaches to modulate kinase activity in gut epithelium (Aim 3). To achieve these aims, during the mentored K99 phase, I will work closely with my mentor Dr. Steven Altschuler and collaborator Dr. Melanie Cobb, recognized experts in single cells analysis and kinase biology respectively, to implement my research and career plan. By taking advantage of exciting advances in intestinal stem cell culture and high-content imaging, this award will enable me to establish a cutting edge research program with the long-term goal of addressing the exquisite control mechanisms of epithelial systems.